The study of tumor suppressor genes (TSGs) has resulted in many important insights into the basic biology of cancer and normal cellular pathways including the cell cycle, apoptosis and DNA repair. RhoBTB2 was recently identified as a candidate TSG located at chromosome 8p21, a site of frequent loss of heterozygosity in breast, lung, prostate and ovarian cancer. RhoBTB2 contains an N-terminal GTPase domain, related to those of Rho family GTPases, and tandem BTB domains. We began investigations to understand the function of RhoBT2 and to determine whether it is a TSG. We identified RhoBTB2 interacting proteins using yeast two hybrid screens. Our initial screen revealed that RhoBTB2 binds to the ubiquitin ligase Cul3. RhoBTB2 is ubiquitinated in a CulS-dependent manner and the cellular levels of RhoBTB2 are regulatedby Cul3-dependent degradation. A RhoBTB2 mutant found in a lung cancer cell line fails to bind to Cul3 and its expression is not regulated by Cul3, indicating that interaction with Cul3 is probably an essential component of the function of RhoBTB2 as a tumor suppressor. These data also suggest that RhoBTB2 may be an adaptor whose primary role is to recruit another protein(s) to Cul3 for degradation. Adaptors for other cullin proteins are also tumor suppressors. We conducted additional two hybrid screens and have identified other interacting proteins that indicate the function of RhoBTB2. Our goals in this proposal are to determine whether RhoBTB2 is a TSG, understand in detail the pathways in which RhoBTB2 functions, and delineate the roles played by Cul3 and ubiquitination in regulating RhoBTB2 function. We plan to achieve these goals through three specific aims. We will: 1) Define the role of RhoBTB2 as a tumor suppressor;2) Determine the function of RhoBTB2;and 3) Delineate the role of Cul3 in RhoBTB2 function. The results of these studies may reveal a new pathway involved in the development of cancer and advance our understanding of the basic biology of transformation.